Previously we demonstrated a biochemical rationale for combination of 5-fluorouracil (FUra) with a reduced folate such as leucovorin (5-CHO-H4PteGlu) in the treatment of adult colon adenocarcinoma. The success of subsequent clinical trials has stimulated evaluation of this combination in other malignancies including osteosarcoma and colon adenocarcinoma of children at SJCRH. However, it is unknown whether a similar rationale exists for pediatric solid tumors, since biochemical determinants of this synergism have not been elucidated for any tumor type. We propose to examine the interaction of leucovorin and FUra in appropriate human models of childhood osteosarcoma and colon adenocarcinoma maintained as xenografts. Our initial studies will determine whether resistance de novo to FUra is a consequence of transient inhibition of thymidylate synthase, and if this is a consequence of low endogenous CH2- H4PteGlu or its polyglutamate forms. Optimal concentrations of ligands (CH2-H4PteGlu, FdUMP) for formation and stability of convalent ternary complex will be established, and compared to ligand concentrations in situ. In mice a relationship between reduced folates in plasma and in tumors during leucovorin infusion will be established. By manipulating i.v. infusion conditions (time and dose) it will be possible to determine whether this has influence on the inhibition of thymidylate synthase, the pools of CH2-H4PteGlu, and distribution of its polyglutamate forms. Such studies will be correlated with tumor response, and complemented by detailed examination of the effect of polyglutamylation in formation and stability of covalent ternary complex using thymidylate synthase purified from an osteosarcoma xenograft. As leucovorin is a racemic mixture of active (S) and inactive (R) isomers, we will attempt in vitro and in vivo to define the influence of the biologically inactive species on metabolism and synergistic interaction of (6S)5-CHO-H4PteGlu with FUra. These studies will a) increase our understanding of drug resistance mechanisms in these childhood tumors b) define biochemical determinants of the FUra-leucovorin interaction, and c) aid in optimizing FUra-leucovorin therapy for overcoming resistance de novo.